Gastrointestinal Neurons Expressing HCN4 Regulate Retrograde Peristalsis

Fujii, Kensuke; Nakajo, Koichi; Egashira, Yasuyuki; Yamamoto, Yoshimitsu; Kitada, Kazuya; Taniguchi, Kohei; Kawai, Masaru; Tomiyama, Hideki; Kawakami, Koichi; Uchiyama, Kazuhisa; Ono, Fumihito

doi:10.1016/j.celrep.2020.02.024

Cited by 14 publications

(12 citation statements)

References 54 publications

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“…While technical issues could be responsible for the presence of those markers, we rather suggest that glial gene expression in ChAT Cre /Rpl22 HA/+ mice is a remnant feature from their shared neural progenitors 31. Furthermore, recent studies in zebra fish gut show an expression of ICC marker Hcn4 in enteric neurons,32 in line with the expression in our ChAT neurons, suggesting a use of this channel in mice EN.Although the RiboTag approach brings significant advantages into the intestinal research field, it has some limitations. Low amounts of tissue or isolation of mRNA from rare cell types might prove difficult, but additional steps, such as sample pooling, canassist acquiring sufficient amounts of RNA.…”

mentioning

confidence: 73%

Application of a RiboTag‐based approach to generate and analyze mRNA from enteric neural cells

Leven

Schneider

Siemens

et al. 2021

Neurogastroenterology Motil

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Background: Transcriptional profiling of specific intestinal cell populations under health and disease is generally based on traditional sorting approaches followed by gene expression analysis. Therein, specific cell populations are identified either by expressing reporter genes under a cell type-specific promotor or by specific surface antigens. This method provides adequate results for blood-derived and tissue-resident immune cells. However, in stromal cell analysis, cellular stress due to digestion often results in degraded RNA. Particularly, ramified cells integrated into the tissue, such as enteric neurons and glial cells, suffer from these procedures.These cell types are involved in various intestinal processes, including a prominent immune-regulatory role, which requires suitable approaches to generate cell-specific transcriptional profiles.Methods: Sox10 iCreERT2 and choline acetyltransferase (ChAT Cre ) mice were crossed with mice labeling the ribosomal Rpl22 protein upon Cre activity with a hemagglutinin tag (Rpl22-HA, termed RiboTag). This approach enabled cellular targeting of enteric glia and neurons and the immediate isolation of cell-specific mRNA from tissue lysates without the need for cell sorting. Key results:We verified the specific expression of Rpl22-HA in enteric glia and neurons and provided gene expression data demonstrating a successful enrichment of either Sox-10 + glial or ChAT + neuronal mRNAs by the RiboTag-mRNA procedure using qPCR and RNA-Seq analysis. Conclusions and inferences:We present a robust and selective protocol that allows the generation of cell type-specific transcriptional in vivo snapshots of distinct enteric cell populations that will be especially useful for various intestinal disease models involving peripheral neural cells.

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confidence: 73%

Application of a RiboTag‐based approach to generate and analyze mRNA from enteric neural cells

Leven

Schneider

Siemens

et al. 2021

Neurogastroenterology Motil

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“…In the brain, HCN channels are the molecular correlate of the hyperpolarization-activated current (I h ) that regulates input resistance, pre- and post-synaptic properties, and rhythmic oscillatory patterns ( Wahl-Schott and Biel, 2008 ). And, currents mediated by HCN channels in the gastrointestinal tract regulate peristalsis and mobility ( Fisher et al, 2018b ; Fujii et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Regulation of HCN Channels by Protein Interactions

et al. 2022

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Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels are key regulators of subthreshold membrane potentials in excitable cells. The four mammalian HCN channel isoforms, HCN1-HCN4, are expressed throughout the body, where they contribute to diverse physiological processes including cardiac pacemaking, sleep-wakefulness cycles, memory, and somatic sensation. While all HCN channel isoforms produce currents when expressed by themselves, an emerging list of interacting proteins shape HCN channel excitability to influence the physiologically relevant output. The best studied of these regulatory proteins is the auxiliary subunit, TRIP8b, which binds to multiple sites in the C-terminus of the HCN channels to regulate expression and disrupt cAMP binding to fine-tune neuronal HCN channel excitability. Less is known about the mechanisms of action of other HCN channel interaction partners like filamin A, Src tyrosine kinase, and MinK-related peptides, which have a range of effects on HCN channel gating and expression. More recently, the inositol trisphosphate receptor-associated cGMP-kinase substrates IRAG1 and LRMP (also known as IRAG2), were discovered as specific regulators of the HCN4 isoform. This review summarizes the known protein interaction partners of HCN channels and their mechanisms of action and identifies gaps in our knowledge.

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“…Therefore, HCN channels are also known as pacemaker channels because they control heart rate by generating pacemaker potentials ( Ludwig et al, 1999 ; Moosmang et al, 2001 ; Brioschi et al, 2009 ). In addition to the heart, HCN channels are expressed in the central nervous system ( Santoro et al, 2000 ) and the gastrointestinal nervous system ( Fujii et al, 2020 ), where they play a role in the regulation of neuronal excitability and synaptic transmission ( Beaumont and Zucker, 2000 ).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, due to the genome duplication in teleost evolution, the zebrafish genome sometimes has two orthologs to one gene in mammals ( Amores et al, 1998 ; Glasauer and Neuhauss, 2014 ). In the case of HCN4, there is only one HCN4 gene in the human genome, HsHCN4 (Hs: Homo sapiens ), whereas the zebrafish genome contains two HCN4 channel genes, DrHCN4 (Dr: Danio rerio ) and DrHCN4L ( Fujii et al, 2020 ; von der Heyde et al, 2020 ). It has been shown that DrHCN4 is expressed in the heart and gastrointestinal tract ( Fujii et al, 2020 ; Stoyek et al, 2022 , 2015 ).…”

Section: Introductionmentioning

confidence: 99%

“…In the case of HCN4, there is only one HCN4 gene in the human genome, HsHCN4 (Hs: Homo sapiens ), whereas the zebrafish genome contains two HCN4 channel genes, DrHCN4 (Dr: Danio rerio ) and DrHCN4L ( Fujii et al, 2020 ; von der Heyde et al, 2020 ). It has been shown that DrHCN4 is expressed in the heart and gastrointestinal tract ( Fujii et al, 2020 ; Stoyek et al, 2022 , 2015 ). Cardiac automaticity in zebrafish utilizes membrane mechanism along with calcium clock mechanisms as in mammals ( Marchant and Farrell, 2019 ; Stoyek et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Two HCN4 Channels Play Functional Roles in the Zebrafish Heart

Liu

Kasuya²,

Zempo³

et al. 2022

Front. Physiol.

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The HCN4 channel is essential for heart rate regulation in vertebrates by generating pacemaker potentials in the sinoatrial node. HCN4 channel abnormality may cause bradycardia and sick sinus syndrome, making it an important target for clinical research and drug discovery. The zebrafish is a popular animal model for cardiovascular research. They are potentially suitable for studying inherited heart diseases, including cardiac arrhythmia. However, it has not been determined how similar the ion channels that underlie cardiac automaticity are in zebrafish and humans. In the case of HCN4, humans have one gene, whereas zebrafish have two ortholog genes (DrHCN4 and DrHCN4L; ‘Dr’ referring to Danio rerio). However, it is not known whether the two HCN4 channels have different physiological functions and roles in heart rate regulation. In this study, we characterized the biophysical properties of the two zebrafish HCN4 channels in Xenopus oocytes and compared them to those of the human HCN4 channel. We found that they showed different gating properties: DrHCN4L currents showed faster activation kinetics and a more positively shifted G-V curve than did DrHCN4 and human HCN4 currents. We made chimeric channels of DrHCN4 and DrHCN4L and found that cytoplasmic domains were determinants for the faster activation and the positively shifted G-V relationship in DrHCN4L. The use of a dominant-negative HCN4 mutant confirmed that DrHCN4 and DrHCN4L can form a heteromultimeric channel in Xenopus oocytes. Next, we confirmed that both are sensitive to common HCN channel inhibitors/blockers including Cs+, ivabradine, and ZD7288. These HCN inhibitors successfully lowered zebrafish heart rate during early embryonic stages. Finally, we knocked down the HCN4 genes using antisense morpholino and found that knocking down either or both of the HCN4 channels caused a temporal decrease in heart rate and tended to cause pericardial edema. These findings suggest that both DrHCN4 and DrHCN4L play a significant role in zebrafish heart rate regulation.

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Gastrointestinal Neurons Expressing HCN4 Regulate Retrograde Peristalsis

Abstract: Highlights d Gastrointestinal neurons expressing HCN4 are serotonergic d Blocking the HCN4 channel function attenuates retrograde peristalsis d Depolarization of neurons expressing HCN4 strengthens retrograde peristalsis d Neurons expressing HCN4 control circular muscles

Cited by 14 publications

References 54 publications

Application of a RiboTag‐based approach to generate and analyze mRNA from enteric neural cells

Application of a RiboTag‐based approach to generate and analyze mRNA from enteric neural cells

Regulation of HCN Channels by Protein Interactions

Two HCN4 Channels Play Functional Roles in the Zebrafish Heart

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